Eutrophication within coastal waters is perceived as the major problem resulting from nutrient pollution, and nitrogen appears to be the main culprit. Bacterial denitrification was for decades considered the only process that could convert inorganic nitrogen forms completely to N2 gas, thus the environmental factors that control denitrification rates in estuarine sediments and waters have been investigated in some detail. These studies demonstrated that up to 50% to 80% of the external nitrogen load delivered to coastal ecosystems could be lost to sediment denitrification dependent in part on the residence time of water in the estuary, leading to a reduction in both cultural eutrophication and nitrogen pollution of the open ocean. However, the discovery of anaerobic ammonium oxidation (anammox) in a wastewater treatment plant by Mulder (1995) identified an alternate pathway for fixed nitrogen loss to the atmosphere as N2 gas. Indeed this process has been found responsible for 24-67% of N loss in marine and coastal sediments, and is now recognized as an important process for the removal of nitrogen in coastal sediments, anoxic ocean waters and estuarine sediments. Thus, the fate of excess nitrogen in estuaries is determined by the microbial-driven nitrogen cycle, and maintenance of efficient denitrifiers and anammox microorganisms. Estuaries also receive and retain other pollutants, including metals and petroleum compounds, introduced from urban, run-off and erosion, effluent discharge, atmospheric deposition, as well as from regular shipping activities and oil spills. The problem of marine pollution due to these contaminants has become a global concern because not only they can have important deleterious effects on marine organisms of the different trophic levels, but also, in the case of metals, can be bioaccumulated and biomagnified through the food-chain leading to hazards to human health. However, the suggestion that these contaminants can directly affect essential biogeochemical processes represents a new perspective of investigation. Interestingly, recent findings from our research team (Magalhães 2007a, 2009a) have shown a pronounce toxic effect of metals (Cd, Cu, Zn, Pb, Cr) on the enzymes responsible for “classical” denitrification but as yet, there is no information about the toxic effect of metals on anammox enzymes and on the effects of PAHs (polycyclic aromatic hydrocarbons) in both N removal biological processes. In this study we propose to examine the effect(s) of PAHs and metals on the two microbially mediated processes of N removal; denitrification and anammox. This study will be conducted in an estuary located in the North of Portugal (River Lima Estuary), which includes a large shipyard, an important commercial sea-port, and a fishing harbour. We will first characterize different sediments collected along the Lima Estuary, in terms of anammox and denitrification activity and contaminants (Cd, Cr, Cu, Pb, Zn and EPA’s 16 priority PAHs) to provide data on the present levels of contamination, and establish potential relationships with the spatial variability of denitrification and anammox activity. Because many environmental and chemical variables are correlated in an estuary, we secondly will perform controlled laboratory experiments using sediment slurries. Here we will test the effects of the above referred metals and PAHs (a mix of EPA’s 16 priority PAHs) on denitrification and anammox activity and on the diversity and abundance of the active denitrifiers and anammox bacteria by using emergent biochemical and molecular microbial techniques. This proposal represents a comprehensive study on the possible effects of metals and PAHs on anammox and denitrification, and it has the scientific goal of providing an unique contribution to our understanding of the ecology of denitrification and anammox in systems impacted with metals and petroleum compounds. The possible impact of these contaminants on nitrogen removal pathways has profound implications for the management of aquatic ecosystems to ameliorate eutrophication (N-removal). In addition, identification of denitrifier and anammox bacteria, resistant to metals and/or PAHs, is of crucial practical importance for future optimization of fixed N removal in wastewater treatment plants using traditional (denitrification) or emerging (anammox) technologies.